LC/MS/MS and GC/MS/MS metabolic profiling of Leontodon hispidulus, in vitro and in silico anticancer activity evaluation targeting hexokinase 2 enzyme

Leontodon hispidulus Boiss is a wild annual plant growing in Egypt. The present study aims for the first time, to evaluate the phytochemical profile of the main secondary metabolites of the optimized ethanolic extract of the plant using Quadrupole Time-of-Flight Liquid chromatography-mass spectrometry and Gas chromatography-mass spectrometry. It also aims to assess the anticancer activity of its different fractions against the prostate carcinoma cell line. Moreover, an in-silico docking study was performed using the Hexokinase-two enzyme. LC-qToF-MS analysis revealed the tentative identification of 36 phenolic compounds including the glycosides of (luteolin, quercetin, kaempferol, apigenin, isorhamnetin, and daidzein), coumarines (esculin, esculetin, and daphnetin), and phenolic acids (chlorogenic, caffeic, quinic, P-coumaric, and rosmarinic). GC–MS/MS analysis revealed the presence of 18 compounds where palmitic acid, myristic acid, alpha-amyrin, and beta-amyrin were the major ones. The cytotoxic activity results revealed that methylene chloride and ethyl acetate fractions showed the highest cytotoxic activity against the PC3 cell line, with IC50 values of 19, and 19.6 μg/ml, respectively. Interestingly, the docking study demonstrated that apigenin-7-O-glucoside, luteolin-7-O-glucoside, kaempferol-3-O-glucuronide, quercetin-4′-O-glucoside, esculin, rosmarinic acid, chlorogenic acid, and α-amyrin exhibited high affinity to the selected target, HEK-2 enzyme.

Leontodon is a rare wild plant of the family Asteraceae.It includes about 50 species that are geographically distributed through the Mediterranean, European, and Asian countries 1 .Leontodon hispidulus Boiss. is a member of the genus Leontodon that grows as a wild plant in Egypt.It is an annual plant, stemless with a rigid taproot, simple glandular hairs, rosette, oblong-oblanceolate leaves, and yellow-orange flowers 1 .Family Asteraceae is well-known for its flowering plants with a wide range of traditional uses.For example, Carduus species have often been used as anti-hemorrhoidal and cardiotonic remedies in traditional medicine, and Onopordum tauricum as a remedy for liver disease.The flowers and roots from Onopordum acanthium were used as antipyretic and diuretic agents, and Centaurea solstitalis is used in folk medicine to treat stomach problems, abdominal pain, herpes infections, and the common cold.Tanacetum parthenium, also known as feverfew in folk medicine and medieval aspirin, has been used as a remedy for headaches, migraine, nausea, vomiting, stomachaches, rheumatism, and other inflammations.Another plant with practical uses is Bidens pilosa, also known as Spanish needles, which grows mostly in subtropical and tropical regions.It has been used as a remedy for liver problems and to lower blood pressure.In addition, Carthamus tinctorius (safflower) is a treatment for rheumatism and osteoporosis in Korean herbal medicine.Cichorium intybus (chicory) is used in traditional medicine as a remedy for inflammation and liver disorders.Tonics from C. intybus have also been used to treat enlarged spleen and fever in Indian Ayurveda medicine, and a decoction from the leaves was used as a cure for rheumatism and gout.Tea prepared from Achillea aleppica and Achillea biebersteinii was recommended for abdominal pain.The aerial parts from Chrysophthalmum montanum were boiled and applied to wounds and other injuries.Matricaria aurea was recommended in the diet twice a day for bronchitis, sore throat, and cough 2 .Sesquiterpenoids especially guaiane-type compounds are the unique chemotaxonomic markers of Leontodon in the flower heads of L. autumnalis and the roots of L. hispidus 3,4 .Hypocretenolide glycoside was first isolated from L. hispidus, which had a potent cytotoxic activity against CD34( +) bone-marrow stem cell malignancy 5 and anti-inflammatory activity 6 .In addition, other secondary metabolites had been reported in Leontodon taxa like phenolic acids (chicoric, chlorogenic, caffeoyl tartaric, and 3,5-dicaffeoyl quinic acids) as well as luteolin and its glycosides 7,8 .Moreover, a novel oleanene triterpene was isolated from the chloroform extract of the aerial parts of L. filii which showed a fungicidal activity 9 .
In our previous work 10 we reported that this plant species contains certain classes of secondary metabolites like sterols, flavonoids, coumarins, tannins, and terpenes through phytochemical screening.The High-performance liquid chromatography analysis showed the presence of chlorogenic acid, p-coumaric acid, rutin, quercetin, and kaempferol in L. hispidulus aerial parts 95% ethanolic extract.We also managed to optimize the plant extraction process of both the yield and total phenolic content by applying the Box-Behnken Design.The optimized extract produced the maximized concentration of phenolics (104.18 ± 0.37 μg/mg) with a minimum percentage of errors (− 0.27%) and flavonoid content (29.73 ± 0.0 μg/mg) using 201 ml of 74.5% ethanol/water at 72 h of extraction.The optimized extract showed good biological activities.The antioxidant activity using 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was found to be 41.89 mg Trolox-equivalent/gm, with 80% free radicals inhibition.Moreover, 100 mg/kg of the extract inhibited the edema in rats by 83.5% after 4 h of carrageenan injection as compared to 81.7% inhibition by indomethacin.The extract also showed good anticancer activity on both prostate carcinoma (PC3) and cervical carcinoma (HELA) cell lines (IC 50 = 16.5 and 23 µg/ml, respectively) 10 .More studies were required to unveil the types of secondary metabolites of L. hispidulus.Prostate carcinoma is the majorly recorded malignancy in men, leading to many complications and finally mortality.Its early detection could be achieved by screening for serum prostate-specific antigen.Despite the advanced research to allow early diagnosis, the onset of the disease is often asymptomatic, it develops slowly and spreads from the prostate to other parts of the body.The standard primary treatment includes radical prostatectomy, radiation, and androgen deprivation therapy 11 .Unfortunately, patients with an initial response to treatment will relapse within three years with androgen-independent prostate cancer (PC3) which is rapidly fatal 12 .Generally, cancer treatment includes chemotherapy, radiotherapy, surgery, the transformation of stem cells, photodynamics, and immunotherapy.Severe side effects result from such treatments as limited bioavailability, toxicity, nonspecificity, fast clearance, and metastasis 13 .There are also many undesirable side effects that cancer patients find difficult to tolerate such as nausea, vomiting, anemia, fatigue, hair loss, appetite changes, constipation, bleeding, and infection.Considering this, there is a global need to look for more selective active compounds of natural origin.Hexokinase-2 enzyme (HEK-2) is an important target that is over-expressed in most human cancers and has been proven to be a promising target for cancer therapy.Although HEK-2 has been a potential target for cancer treatment for many years, several challenges remain in the discovery and design of efficient and selective HEK-2 inhibitors.These challenges arise out of the structural properties of HEK-2, including the high polarity of the active site of the enzyme and the difficulty in specifically inhibiting the different isoenzymes 14 .Computational or in-silico docking studies are important to assess the drug-like properties of lead compounds to predict their biological activity in-vivo.These studies help in the fast prediction of relevant properties and important parameters that should be met by a compound for it to be considered as a potential drug candidate.This objective is achieved by the application of automated software which can be used for making various predictions.Drug uptake, its absorption, evacuation, and associated hazardous effects are important factors for consideration in drug design and should be known in the early stages of drug development.Several important physicochemical properties like molecular weight, polar surface area, molecular flexibility, etc. have to be taken into consideration in drug designing.Toxicological assessment is another important aspect of drug discovery which predicts the safety and adverse effects of a drug.Therefore, bioactivity scores of probable drug leads against various human receptors can be used to predict and evaluate the probability of them acting as potential drug candidates in-vivo 15 .
The study aims to identify the major active compounds in L. hispidulus optimized extract, including phenolic compounds and terpenes, evaluate the cytotoxic activity of its successive fractions against prostate carcinoma cell lines (PC3), and test the inhibitory effect of the main compounds against HEK-2 enzyme in-silico.

Materials Plant material collection and identification
The aerial parts of Leontodon hispidulis Boiss.were collected in March 2022 at the flowering stage from the north coast of Egypt, it was kindly identified by Dr. Soad Abdallah Hassan, Professor of flowering plants, Botany Department, Faculty of Science, Ain Shams University, Cairo, Egypt.Voucher specimens of the plant was deposited in the herbarium of "Pharmacognosy department, Faculty of Pharmacy, Cairo University, Cairo, Egypt" with the code number (21-3-17).Plant collection and experimental protocol were achieved after permission from the "Desert Research Center, Cairo, Egypt", and the "Ethical Committee of the Faculty of Pharmacy, Cairo University, Cairo, Egypt" (serial number of the protocol: MP 1841).Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, must comply with relevant institutional, national, and international guidelines and legislation.

Chemicals
Solvents for extraction and fractionation Ethanol, petroleum ether, methylene chloride, ethyl acetate, butanol (all HPLC grade) were purchased from Sigma-Aldrich, Germany.

LC-qTOF-MS of the total extract
Mobile phase (negative mode): 5 mM ammonium formate buffer (pH = 8) containing 1% methanol.All solvents were HPLC grade and were kindly provided and prepared by Proteomics Laboratory of Children Cancer Hospital 57357, Cairo, Egypt.

GC-MS/MS of the petroleum ether fraction
Solvent: petroleum ether (HPLC grade) was purchased from Sigma Aldrich Chemicals, Germany.
Mobile phase: Helium gas at a flow rate of 1.0 ml/min at a splitless mode was provided by Central Laboratories Network, National Research Center, Giza, Egypt.

Cytotoxic activity
Cell lines: Human prostate carcinoma (PC3) cell lines were purchased from the National Cancer Institute, Cairo, Egypt.Doxorubicin: as a standard anticancer drug, it was purchased from Sigma Aldrich Chemicals, Germany.

Preparation of the plant material
The powder of the aerial parts was extracted by maceration at room temperature using 201 ml of 74.5% ethanol/ water at 72 h for 20 g powder to obtain the optimized extract with the maximum phenolic content 10 .The solvent was evaporated using a Rotatory Evaporator "BuchiRR-300, USA" at 45°.The resulting residue was subjected to fractionation with solvents of increasing polarity (petroleum ether, methylene chloride, ethyl acetate, butanol, and water).

LC-qTOF-MS of the total extract
The separation process was performed using a Shimadzu UPLC system (Kyoto, Japan) equipped with a binary solvent delivery system and an autosampler, coupled with ACQUITY UPLC BEH-C18 (150 × 2.1 mm, 1.7 um: Waters, USA).The column temperature was maintained at 25 °C and mass spectrometry was performed on a Triple TOF™ 5600 + system with a Duo-Spray™ source operating in the Negative Electron Spray Ionization mode (ESI) (AB SCIEX, CA, USA).The flow rate was kept at 0.3 ml/min.The mobile phase A (5 mM ammonium formate buffer (pH = 8) containing 1% methanol) and mobile phase B (100% acetonitrile) were applied according to the gradient elution program demonstrated in Table 1.

Sample preparation for LC-MS/MS analysis
The optimized 74.5% ethanol/water extract of L. hispidulus was used in the LC-MS/MS analysis 10 .One ml mobile phase working solution (MP-WS) [DI-Water: Methanol: Acetonitrile-50: 25: 25] was added to 50 mg sample, followed by vortex for 2 min, then ultra-sonication for 10 min and centrifuge for 10 min at 10,000 rpm.20 µl stock (50/1000 µl) was diluted with 1000 µl reconstitution solvent.Finally, the injected concentration was 1 µg/ µl.The injection volume was 10 µl and 10 µl MP-WS as a blank.

Data processing
Respect Negative (1573 records) was the database used 16 .MasterView Program was used for feature (peaks) extraction from the Total ion chromatogram (TIC) based on the following criteria; features should have Signalto-Noise > 5 (Non-targeted analysis), features intensities of the sample-to-blank > 5.
The following optimized parameters were applied; ion spray voltage (5.5 kV), ion source heater (550 °C), curtain gas [35 psi-gas 1 (nebulizer gas, 55 psi), gas 2 (heater gas, 55 psi)], declustering potential (60 eV), collision energy (30 eV), collision energy spread (15 eV).The scan range was from 50 to 800 m/z with a 250 ms accumulation time.An automated calibration delivery system (CDS) was applied for the regulation of the MS and the MS/MS automatically.

GC-MS/MS of the petroleum ether fraction
The GC-MS/MS system (Agilent Technologies) was equipped with a gas chromatograph (7890B) and mass spectrometer detector (5977A) at Central Laboratories Network, National Research Centre, Giza, Egypt.The GC was equipped with HP-5MS column (30 m × 0.25 mm internal diameter and 0.25 μm film thickness).The petroleum ether fraction was subjected to a silylation process using 1% trimethylchlorosilane (TMCS).Helium was the carrier gas (1.0 ml/min flow rate) in a splitless mode and the injection volume was (0.1 µl).The following temperature program was applied [80 °C for 2 min; rising at 5 °C/min to 300 °C and held for 10 min].The injector and detector were held at 280 °C and 300 °C, respectively.Mass spectra were obtained by electron ionization (EI) at 70 eV; using a spectral range of m/z 25-550 and solvent delay of 3.7 min.Identification of different constituents was determined by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.

In-vitro cytotoxic activity
Measurement of the potential cytotoxic activity was performed on prostate carcinoma cell lines (PC3), using the successive fractions of the optimized ethanolic extract of L. hispidulus [petroleum ether, methylene chloride, ethyl acetate, butanol, and aqueous fractions], while doxorubicin was used as a positive control.The procedures were www.nature.com/scientificreports/according to the Sulforhodamine-B assay (SRB) 10 .Each value is the mean of three replicates.Obtained values were presented as mean ± SD.Significant differences between the values were calculated using SPSS software (V.22) using One-way ANOVA and Tukey's test.The difference is considered significant at (P ˂ 0.05).

In-silico docking study using hexokinase-2 enzyme
The molecular modeling studies were carried out using Molecular Operating Environment (MOE, 2019.0102)software.The X-ray crystallographic structure of Hexokinase-2 complexed with the reference compound [2-amido-6-benzenesulfonamide glucosamine derivative (PDB ID: 5HFU)] was downloaded from the protein data bank (https:// www.rcsb.org/ struc ture/ 5HFU).All the tested compounds were compared to the reference compound.

Identification of flavonoids and their glycosides
Different classes of flavonoids and their glycosides were identified in the LC-qTOF-MS spectrum of L. hispidulus optimized ethanolic extract including flavones, flavonols, isoflavones, and flavanones.
Flavonols and their glycosides.Quercetin (flavon-3-ol) was tentatively identified by its molecular ion (m/z = 301) 28 .Two quercetin glycosides were tentatively identified.The first one was quercetin-4ˋ-O-glycoside which was identified by its molecular ion (m/z = 463) and the fragment ion of the aglycone (m/z = 301) after loss of hexose molecule 29 .The other one was quercetin-3-D-xyloside and it was characterized by its molecular ion (m/z = 433) and the same peak of the aglycone (m/z = 301) 30 .Four kaempferol glycosides were tentatively

GC-MS/MS of the petroleum ether fraction of L. hispidulus extract
Table 3 summarizes the identified compounds in L. hispidulus petroleum ether fraction by GC-MS/MS analysis.
The total ion chromatogram is demonstrated in Fig. 3. Figure 4a-g demonstrates the MS/MS spectrum figures of the major identified compounds.   .Guaiane-type sesquiterpenoid compounds were discovered previously in the flower heads of L. autumnalis 3 and the roots of L. hispidus 4 .Moreover, a hypocretenolide glycoside was isolated from L. hispidus, which had a potent cytotoxic activity 5 and anti-inflammatory activity 6 .

Cytotoxic activity of L. hispidulus successive fractions against Prostate carcinoma (PC3)
Results of the cytotoxic study are demonstrated in Figs. 5 and 6, where IC 50 values of the different fractions (petroleum ether, methylene chloride, ethyl acetate, butanol, and aqueous) against prostate carcinoma were (23.5, 19, 19.6, 28.1, and 21.1 μg/ml, respectively).The most active fractions were methylene chloride and ethyl acetate with very close IC 50 values (19 and 19.6 μg/ml, respectively), while the standard doxorubicin IC 50 was 5.1 μg/ml.In our previous work 10 , the IC 50 of L. hispidulus total optimized ethanolic extract against the PC3 cell line was 16.5 μg/ml which was more potent anticancer activity than each fraction alone.This result indicates a synergistic activity between the five fractions.Statistical analysis of the data of the surviving fractions was performed by SPSS software (V.22) using One-way ANOVA and Tukey's test.Table 4 represents The One-way ANOVA results for the surviving fractions of Leontodon hispidulus Boiss.comparing the successive fractions to doxorubicin.Table 5 demonstrates The Multiple Comparison: Post Hoc Test-Tukey's HSD results.Interestingly, both methylene chloride and ethyl acetate fractions showed a non-significant difference from the standard doxorubicin through the lowest concentrations (05.00, 12.50, and 25.00 µg/ml), while the difference was significant (higher in activity than doxorubicin) at the highest concentration (50.00 µg/ml).As demonstrated in Table 5, group (2) [methylene chloride fraction] showed a non-significant difference from group (6) [doxorubicin] through the first three concentrations [05.00 µg/ml, P-value = 0.077, 12.50 µg/ml, P-value = 0.319] and [25.00 µg/ml, P-value = 0.976].

In-silico docking study using hexokinase-2 enzyme
The structural properties of HEK-2 represent a challenge due to the high polarity of the active sites of the enzyme and the difficulty in specifically inhibiting the different isoenzymes 14 .Therefore, eight identified compounds of varying polarity were selected for the in-silico docking study.The molecular modeling study was carried out using Molecular Operating Environment (MOE, 2019.0102)software.The docking setup was first validated by self-docking of the co-crystallized ligand in the vicinity of the binding site of the enzyme, the docking score (S) was − 8.018 kcal/mol.and root mean square deviation (RMSD) was 1.931 Å Figs. 7 and 8.As demonstrated in (Table 6), all the tested eight compounds showed promising inhibitory effects on the HEK-2 enzyme.Apigenin-7-O-glucoside (Fig. 9) was the most active compound with a binding score of -7.412 through three hydrogen bonds with HEK-2.Two bonds as a H-bond donor with Asp657 and Glu742 amino acid residues and  one bond as a H-bond acceptor with Lys621.luteolin-7-O-glucoside (Fig. 10) was found to be the second most active compound with a very close score of − 7.342.IT also formed three H-bonds with HEK-2, two as a H-bond donor with the amino acid residues Asp657 and Glu708, and one as a H-bond acceptor with Asn656.There were also two additional H-π interactions with Cys606 and Pro605 amino acid residues.Both compounds proved to possess an anticancer activity 44 .This result was in good agreement with the previous study of Palombo 45 and a previous study on L. saxatilis 43 that reported the anticancer activity of both apigenin 4′-O-β-D-glucoside, luteolin 7-O-β-D-glucoside.Kaempferol-3-O-glucuronide (Fig. 11) and quercetin-4′-O-glucoside (Fig. 12) were next in activity due to the similar structure to the first two compounds.Kaempferol-3-O-glucuronide showed anticancer activity using the Ehrlich ascites assay 46 , while quercetin-4′-O-glucoside proved its anticancer potential against hepatoblastoma cell line (HepG-2), PC3 and colorectal adenocarcinoma (HT-29) cell lines 47 .Importantly, esculin (Fig. 13) showed a high binding affinity with a score of -6.210 despite being smaller in size than the previous flavonoid glycosides.It formed three H-bonds as a H-bond donor, one H-bond as a H-bond acceptor, and one H-π interaction.Esculin was reported to have good anticancer activity via induction of apoptosis and autophagy in human glioblastoma multiforme cells (T98G) and human anaplastic astrocytoma cells 48 .Rosmarinic acid (Fig. 14) came very close to esculin with a score of − 6.144 through the formation of two H-bonds as a H-bond donor, one as a H-bond acceptor, and one as H-π interaction.Rosmarinic acid showed anticancer activity against many types of cancer as colon, skin, lung, and ovarian cancers 49 .Finally, came chlorogenic acid (Fig. 15) and the triterpene alpha-amyrin (Fig. 16) with scores of − 5.616 and − 5.272, respectively.Chlorogenic acid proved its activity against breast carcinoma 50 , while alpha-amyrin was reported to have anticancer activity against www.nature.com/scientificreports/hepatocellular carcinoma via an in-silico docking study 51 .This result may explain the promising synergistic anticancer activity of the different fractions of L. hispidulus optimized extract against prostate carcinoma cell lines.

Conclusion
The comprehensive profiling of L. hispidulus optimized ethanolic extract revealed the presence of thirty-six phenolic compounds which were identified for the first time in this plant species using LC-qTOF-MS.These compounds include the glycosides of (luteolin, quercetin, kaempferol, apigenin, isorhamnetin, and daidzein), coumarines (esculin, esculetin and daphnetin), phenolic acids (chlorogenic, caffeic, quinic, P-coumaric and rosmarinic).GC-MS/MS also was performed for the first time revealing the presence of eighteen fatty acids and hydrocarbons as palmitic acid, myristic acid, α-linolenic acid, hexadecanoic acid, oleic acid, behenic acid, and triterpenes (α-and β-amyrin).Methylene chloride and ethyl acetate were the most active fractions against the PC3 cell line.They showed very close IC 50 values (19 and 19.6), respectively.It is worthy to mention, that all the successive fractions (except for the butanol fraction) showed a significantly higher anticancer activity against the PC3 cell line than doxorubicin at the highest concentration (50.00 µg/ml).Apigenin-7-O-glucoside showed the highest inhibitory effect of HEK-2 enzyme with a binding score of − 7.412, followed by luteolin-7-O-glucoside, kaempferol-3-O-glucuronide, quercetin-4ˋ-O-glucoside, esculin, rosmarinic acid, chlorogenic acid and finally alpha-amyrin.The results of the docking study showed consistency with the results of the anticancer activity study on the successive fractions, as the synergistic activity could be due to the flavonoid glycosides, coumarins, phenolic acids, and triterpenes combined.This study could be a starting point for many future studies on L. hispidulus to further investigate its biological potential.

Figure 2 .
Figure 2. (continued) L. hispidulus petroleum ether fraction.Trimethylsilyl derivatives of the fatty acids show a characteristic fragmentation pattern.Hexadecanoic acid TMS derivative (palmitic acid) is the major saturated fatty acid that exists.A characteristic peak appears at m/z = 313 [M-15] due to the loss of methyl group from the silyl part which makes the cation more stable.A characteristic peak appears at m/z = 145 [(CH 3 ) 3 SiCO 2 C 2 H 4 ] + due to the cleavage of the β, γ bond (relative to the carbonyl group).Another characteristic peak appears at m/z = 132 [(CH 3 ) 3 SiCO 2 CH 3 ] + due to Mclafferty rearrangement.Generally, in TMS derivatives, the peak m/z = 73 [CH 3 ) 3 Si] + is the base peak.Another important peak appears at m/z = 117 [(CH 3 ) 3 SiCO 2 ] + .A similar fragmentation pattern applies to other saturated TMS derivatives of fatty acids as myristic acid41 .9,12-Octadecadienoic acid TMS derivative is the main unsaturated fatty acid detected.
The peak m/z = 337 [M-15] appears due to the loss of methyl group from the silyl part.The peak m/z = 73 [CH 3 ) 3 Si] + is the base peak.The compound follows a similar fragmentation pattern to the saturated fatty acids except that the peaks at m/z = 132 [(CH 3 ) 3 SiCO 2 CH 3 ] + and at m/z = 145 [(CH 3 ) 3 SiCO 2 C 2 H 4 ] + are less prominent 41 .Two triterpenes were detected for the first time in the genus Leontodon, α-amyrin (ursane-type) and β-amyrin (oleanane-type).Both follow the Retro Diels-Alder fragmentation pattern.Similar to the fatty acids, the base peak is also at m/z = 73 [CH 3 ) 3 Si] + for both α-and β-amyrin.Also, the peak at m/z = 483 [M-15] due to the loss of methyl group from the silyl part appears in both compounds.The peak at m/z = 218 is prominent in both which is characteristic of Retro Diels-Alder fragmentation.The peak at m/z = 189 is characteristic of α-amyrin, while the peak at m/z = 216 is indicative of β-amyrin

Figure 3 .
Figure 3. Chromatogram of the GC/MS of the petroleum ether fraction of Leontodon hispidulus Boiss.

Figure 7 .
Figure 7. 2D interactions of the native ligand within the hexokinase-2 active site.

Figure 8 .
Figure 8. 3D representation of the superimposition of the co-crystallized (green) and the docking pose (red) of the native ligand in the active site of hexokinase-2.

Table 2 .
Tentative identification of the major secondary metabolites of Leontodon hispidulus Boiss.optimizedextract in the negative mode.Peak no.Peak number, Rt (min) Retention time in minutes, M-H (m/z) Mass divided by Charge number in the Negative mode, MS/MS Mass fragmentation.Peak no.Rt (min) M-H m/z Molecular formula Error of m/z Ms/Ms

Table 3 .
The identified compounds in Leontodon hispidulus Boiss.petroleum ether fraction by GC-MS/MS analysis.Peak no.Peak number, Rt (min) Retention time in minutes, MS/MS Mass fragmentation.

Table 4 .
The One-way ANOVA results for the surviving fractions of Leontodon hispidulus Boiss.comparing the successive fractions to doxorubicin.*The mean difference is significant at (P < 0.05).Groups: petroleum ether, methylene chloride, ethyl acetate, butanol, aqueous and doxorubicin.